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PubMed | Babraham Institute and The Medical Research Council Mitochondrial Biology Unit
Type: Journal Article | Journal: Open biology | Year: 2015

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the -proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a inhibitor protein, which binds to the catalytic F-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.


Narendra D.,U.S. National Institutes of Health | Narendra D.,The Medical Research Council Mitochondrial Biology Unit | Walker J.E.,U.S. National Institutes of Health | Walker J.E.,The Medical Research Council Mitochondrial Biology Unit | Youle R.,U.S. National Institutes of Health
Cold Spring Harbor Perspectives in Biology | Year: 2012

Mutations in Parkin or PINK1 are the most common cause of recessive familial parkinsonism. Recent studies suggest that PINK1 and Parkin form a mitochondria quality control pathway that identifies dysfunctional mitochondria, isolates them from the mitochondrial network, and promotes their degradation by autophagy. In this pathway the mitochondrial kinase PINK1 senses mitochondrial fidelity and recruits Parkin selectively to mitochondria that lose membrane potential. Parkin, an E3 ligase, subsequently ubiquitinates outer mitochondrial membrane proteins, notably the mitofusins and Miro, and induces autophagic elimination of the impaired organelles. Herewe reviewthe recent rapid progress in understanding the molecular mechanisms of PINK1- and Parkin-mediated mitophagy and the identification of Parkin substrates suggesting how mitochondrial fission and trafficking are involved. We also discuss how defects in mitophagy may be linked to Parkinson's disease. © 2012 Cold Spring Harbor Laboratory Press; all rights reserved.


PubMed | The Medical Research Council Mitochondrial Biology Unit
Type: Journal Article | Journal: Biochimica et biophysica acta | Year: 2016

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the electron transport chain in mammalian mitochondria. Extensive proteomic and structural analyses of complex I from Bos taurus heart mitochondria have shown it comprises 45 subunits encoded on both the nuclear and mitochondrial genomes; 44 of them are different and one is present in two copies. The bovine heart enzyme has provided a model for studying the composition of complex I in other mammalian species, including humans, but the possibility of additional subunits or isoforms in other species or tissues has not been explored. Here, we describe characterization of the complexes I purified from five rat tissues and from a rat hepatoma cell line. We identify a~50kDa isoform of subunit NDUFV3, for which the canonical isoform is only ~10kDa in size. We combine LC-MS and MALDI-TOF mass spectrometry data from two different purification methods (chromatography and immuno-purification) with information from blue native PAGE analyses to show the long isoform is present in the mature complex, but at substoichiometric levels. It is also present in complex I in cultured human cells. We describe evidence that the long isoform is more abundant in both the mitochondria and purified complexes from brain (relative to in heart, liver, kidney and skeletal muscle) and more abundant still in complex I in cultured cells. We propose that the long 50kDa isoform competes with its canonical 10kDa counterpart for a common binding site on the flavoprotein domain of complex I.


PubMed | The Medical Research Council Mitochondrial Biology Unit and The Medical Research Council Laboratory of Molecular Biology
Type: | Journal: Proceedings of the National Academy of Sciences of the United States of America | Year: 2016

The structure of the intact monomeric ATP synthase from the fungus, Pichia angusta, has been solved by electron cryo-microscopy. The structure provides insights into the mechanical coupling of the transmembrane proton motive force across mitochondrial membranes in the synthesis of ATP. This mechanism requires a strong and integral stator, consisting of the catalytic


PubMed | The Medical Research Council Mitochondrial Biology Unit
Type: Comparative Study | Journal: The Biochemical journal | Year: 2015

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl--D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl--maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.


PubMed | National Autonomous University of Mexico, The Medical Research Council Mitochondrial Biology Unit and The Medical Research Council Laboratory of Molecular Biology
Type: Journal Article | Journal: Acta crystallographica. Section F, Structural biology communications | Year: 2015

The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the -proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the inhibitor protein, an -helical protein that binds to the catalytic F1 domain of the enzyme. This domain is a complex of three -subunits and three -subunits, and one copy of each of the -, - and -subunits. Attempts to crystallize the F1- inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the - and -subunits only. Its structure was determined to 2.3 resolution and consists of a heterodimer of one -subunit and one -subunit. It has no bound nucleotides, and it corresponds to the `open or `empty catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized.


PubMed | The Medical Research Council Mitochondrial Biology Unit
Type: Journal Article | Journal: Journal of molecular biology | Year: 2011

In the structure of bovine F(1)-ATPase inhibited with residues 1-60 of the bovine inhibitor protein IF(1), the -helical inhibitor interacts with five of the nine subunits of F(1)-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF(1) destabilizes the interaction of the inhibitor with F(1)-ATPase and may assist in removing the inhibitor from its binding site when F(1)F(o)-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long -helix of IF(1) and the C-terminal domains of the (DP)-subunit and (TP)-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the (DP)-subunit. Several conserved charged amino acids in the long -helix of IF(1) are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F(1)-ATPase and occupy aqueous cavities in F(1)-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120 steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme.

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